Synthesis of silica particles and their application as supports for alcohol dehydrogenases and cofactor immobilizations: Conformational changes that lead to switch in enzyme stereoselectivity

FTIR, circular dichroism (CD) and fluorescence spectroscopies were used to characterize conformational changes in horse liver alcohol dehydrogenase (HLADH) and ketoreductase (KRED 117) upon physical and covalent immobilizations on silica particles (functionalized with amino, epoxy and thiol groups) of different sizes. Conformational changes for immobilized enzymes were associated with high and low frequency shifts of the amide I and II bands. CD spectra of native HLADH and KRED 117 characterized with a negative peak at 222 nm indicating a α-helical structure. The disappearance of the negative peak in the CD spectra of immobilized enzymes and appearance of a positive peak at 222 nm supported these observations. These findings demonstrated unfolding of folded enzymes and exposure of the amino acid residues during denaturation with a red shift in tryptophan fluorescence. The decrease in specific activities (by 60–70% in all cases) for both immobilized enzymes was correlated to those of conformational changes. Silica-attached enzyme-NADH systems were evaluated for enantioselective reduction of 1-(p-methoxyphenyl)-propan-2-one. Conformational changes enhanced the enantioselectivity of immobilized HLADH with a switch in its stereoselectivity. In the case of immobilized KRED 117, kinetic values (Vmax and Km) were lower than that of the free enzyme, without enhancing enzyme enantio- and stereoselectivity.

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► Alcohol dehydrogenases and cofactor were immobilized on silica particles.
► Frequency shift of amide I and amide II vibrational bands of immobilized enzymes.
► Changes in enzymes secondary and tertiary structures during immobilization.
► Switch in stereoselectivity of immobilized enzyme.